Tumor necrosis factor alpha (TNF-α) inhibiting pharmaceuticals

ABSTRACT

Pharmaceutical compositions are described for preventing TNF toxicity, comprising as active ingredient the stereospecific (+) enantiomer having 3S, 4S configuration of delta 6 tetrahydrocannabinol type compounds. The compositions are particularly effective in alleviating and even preventing neurotoxicity due to elevated levels of TNF, including septic shock, cachexia and trauma. They are also effective in the treatment of certain chronic degenerative diseases characterized by TNF production, including autoimmune diseases.

This is a continuation of application Ser. No. 08/952,660, filed Nov.17, 1997 now issued U.S. Pat. No. 5,932,610

FIELD OF THE INVENTION

This present invention relates to the use of pharmaceutical compositionsfor inhibiting the production or blocking the action of Tumor NecrosisFactor α (TNF-α), and for preventing or alleviating diseases andconditions associated with this cytokine, such as septic shock orcachexia. Said pharmaceutical compositions comprise as their activeingredient the stereospecific (+) enantiomers, having (3S,4S)configuration, of Δ⁶-tetrahydrocannabinol (THC) type compounds ofgeneral formula (I), as defined hereinbelow.

BACKGROUND OF THE INVENTION

Tumors necrosis factor alpha (TNF-α) is a pleiotropic cytokine, whichhas been implicated in inflammatory and immunological responses, as wellas in pathogenesis of endotoxic and septic shock (reviewed by Tracey andCerami, Ann. Rev. Med. 45, 491-503, 1994; Glauser et al. Clin. InfectDis. 18, suppl. 2, 205-216, 1994). TNF is one of several cytokinesreleases mainly by mononuclear phagocytic cells in response to variousstimuli. Though the role of cytokines in pathophysiological states hasnot been fully elucidated, it appears that TFN-α is a major mediator inthe cascade of injury and morbidity.

Among the serious disease states related to the production of TNF-α, apartial list includes the following: septic shock; endotoxic shock;cachexia syndromes associated with bacterial infections (e.g.,tuberculosis, meningitis), viral infections (eg., AIDS), parasiteinfections (e.g., malaria), and neoplastic disease; autoimmune disease,including some forms of arthritis (especially rheumatoid anddegenerative forms); and adverse effects associated with treatment forthe prevention of graft rejection.

Septic shock is an often lethal syndrome associated with the massiverelease of host cytokines due to stimuli present on, or released by,invasive micro-organisms. These invasive stimuli induce polyclonalstimulation of the infected host immune system, and include bothlipopolysaccharide (LPS), an endotoxin that stimulates B-cells andmacrophages, and superantigens which are exotoxins that stimulateT-cells.

Septic shock has been recognized generally as a consequence ofgram-negative bacterial infection, but it is now clear that it can alsoresult from infection with gram positive micro-organisms and probablyalso by fungi, viruses and parasites. The microorganism itself, itscomponents or products trigger the host cells, especially themacrophages, to release inflammatory mediators such as TNF-α, therebyinitiating a cascade of events leading to cachexia, sepsis syndrome andseptic shock. TNF-α is a major mediator initiating septic shock, andtherefore stands out as a potential therapeutic target Lynn and Cohen,Clin. Infect. Dis. 20, 143-158, 1995)

Despite vast improvements in intensive care and antibiotic therapy,septic shock remains associated with a very high rate of mortality (30to 90%). The poor prognosis for this syndrome is due to the fact thatthis severe complication of infection results in multiple organ failure,even when the actual infection itself is successfully treated. It is,therefore, apparent that effective therapies for this syndrome are anunmet medical need.

Various therapies have been suggested for the treatment of septic shocksyndrome, but as yet none of these has proven to be clinicallyefficacious. Antibodies against TNF-α prevent the detrimental effects ofsuperantigen (Miethke et al., J. Exp. Med. 175, 91-98, 1992) or LPS(Beutler et al., Science 229, 869-871, 1985). The use of anti-TNFantibodies to treat septic shock is disclosed for example in WO 92/16553(Centocor Inc.). WO 92/01472 (Celltech Ltd.) discloses a multivalentimmunoglobulin used to treat diseases associated with elevated cytokinelevels. Various cytokines that inhibit TNF secretion can also reduce thetoxicity of LPS action (Tzung et al., Eur. J. Immunol. 22, 3097-3101,1992; Gerard et al., J. Exp. Med. 177, 547-550, 1993).

Soluble forms of the TNF binding protein (TBP) (Nophar et al. EMBO J.,9, 3269-3278, 1990) may prevent the action of TNF by preventing bindingto its receptors.

Specific classes of compounds have been suggested for the treatment ofdiseases associated with elevated TNF or other inflammatory mediators,as disclosed for example in WO 95/11014 (Searle & Co.); WO 95/09627, WO95/09624 and WO 95/09623 (SmithKline Beecham Corp.); WO 95/09619(Wellcome Found.); WO 95/03051 (Pharmacia AB); WO 95/01980 (PfizerInc.); EP 629401 (Bayer AG); WO 93/14082 (SmithKline Beecham Corp.); andWO 89/05145 (Hoechst Roussel Pharm Inc.).

None of these disclosures is relevant to the present invention, whichdeals with a class of compounds developed as non-psychotropic analogs oftetra- hydrocannabinol (THC), the active ingredient of marijuana. Someof the compounds of general formula (I) are disclosed in U.S. Pat. Nos.4,179,517 and 4,876,276 and 5,284,867. As disclosed in said U.S.patents, these essentially pure synthetic (+)-(3S,4S)-THC derivativesand analogues are devoid of any undesired cannabimimetic psychotropicside-effects. These known compounds have been described as havinganalgesic, antiemetic, antiglaucoma and neuroprotective activity.

According to the present invention, it is now disclosed that the saidknown compounds, as well as some novel compounds, in addition to havingsaid analgesic, antiemetic, neuroprotective and anti-glaucoma activity,are unexpectedly also effective against the diseases and conditionsmentioned above, by virtue of their ability to block the production oraction of TNF-α.

SUMMARY OF THE INVENTION

The present invention provides pharmaceutical compositions for reducingand even preventing morbidity and mortality associated with theproduction of TNF-α, or other cytokines. The present compositions arealso effective in alleviating other cytokine induced damage includingthe wasting or cachexia associated with AIDS, tuberculosis, neoplasia ortrauma, and may prevent or ameliorate other disease states associatedwith the production of cytokines, including malaria and parasiticinfections.

The compositions of the present invention are also effective in thetreatment of certain chronic degenerative diseases including arthritisand other autoimmune afflictions which are characterized by productionof TNF-α. In this connection, the compositions of the present inventionare contemplated as therapeutically effective in the treatment ofmultiple sclerosis.

The present invention relates to pharmaceutical compositions for thepurposes set out above, in which the active ingredient is a compound ofthe general formula I:

having the (3S,4S) configuration and being essentially free of the(3R,4R) enantiomer,

wherein A - - - - - B indicates an optional 1(2) or 6(1) double bond,

R is

(a) —Q wherein Q is a heterocyclic moiety having a labile hydrogen atomso that said moiety acts as a carboxylic acid analogue,

(b) —R′X wherein R′ is C₁-C₅ alkyl and X is halogen, —OR″ wherein R″ ishydrogen, C₁-C₅ alkyl, or —OC(O)R′″ wherein R′″ is hydrogen or C₁-C₅alkyl,

(c) —R′N(R″)₂ wherein R′ is C₁-C₅ alkyl and each R″, which may be thesame or different, is hydrogen or C₁-C₅ alkyl optionally containing aterminal —OR′″ or —OC(O)R′″ moiety wherein R′″ is hydrogen or C₁-C₅alkyl,

(d) —R′ wherein R′ is C₂-C₅ alkyl,

(e) —R′OR′″ wherein R′ is C₁-C₅ alkyl and R′″ is hydrogen or C₁-C₅alkyl, or

(f) —R′—C(O)OR′″, wherein R′ may be absent and R′ and R′″ are as definedabove;

G is (a) halogen, (b) C₁-C₅ alkyl, or (c) —OR₁ wherein R₁ is (a′) —R″,wherein R″ is hydrogen or C₁-C₅ alkyl optionally containing a terminal—OR′″ or —OC(O)R′″ moiety wherein R′″ is hydrogen or C₁-C₅ alkyl, or(b′) —C(O)R′″ wherein R′″ is as previously defined; and

R₂ is (a) C₁-C₁₂ alkyl, (b) —OR″″, in which R″″ is a straight chain orbranched C₂-C₉ alkyl which may be substituted at the terminal carbonatom by a phenyl group, or (c) —(CH₂)_(n)OR′″ wherein n is an integer of1 to 7 and R′″ is hydrogen or C₁-C₅ alkyl.

In a currently preferred group of compounds, R² designates a1,1-dimethylalkyl radical or a 1,2-dimethylalkyl radical with a total ofat least 7 carbon atoms. Also preferred are precursors of suchcompounds. Particularly preferred compounds are those wherein R² is1,1-dimethylheptyl or 1,2-dimethylheptyl. It is these embodiments of R²that are found in THC and its analogues. However, for thecytokine-inhibiting activity that characterizes the present invention,it is believed that any lower or mid-range alkyl substituent will besuitable at this position.

The compositions of the present invention are particularly effective inalleviating and even preventing morbidity and mortality associated withthe production of TNF-α, or other cytokines. Methods are provided fortreatment of diseases associated with the production of TNF-α byadministering to a patient in need thereof a composition comprising asactive ingredient a therapeutically effective amount of a compound ofgeneral formula I.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the attenuating effect of HU-211 on TNF activity (FIG. 1A)and in reducing TNF levels in the serum of mice (FIG.1B) following theadministration of endotoxin, LPS.

FIG. 2 shows the protective effect of HU-211 in preventing mortality ofmice following the administration of LPS alone, (FIG. 2A) or followingits administration to galactose-amine-sensitized mice (FIG. 2B).

FIG. 3 shows the beneficial effect of HU-211 on blood pressure (FIG. 3a)and on the hematocrit (FIG. 3b) of rats, following LPS administration.

FIG. 4 shows the beneficial effect of HU-211 on the clinical outcome(A), on TNF levels (B) and on cerebral edema (C) in rat brain afterclosed head injury. The effects of pentoxifylline are shown forcomparison (B,C).

FIG. 5 shows the beneficial effect of HU-211 in collagen inducedarthritis in rats.

FIG. 6 shows the improved clinical outcome in experimental allergicencephalomyelitis in rats treated with HU-211.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides pharmaceutical compositions for reducingand even preventing morbidity and mortality associated with theproduction of TNF-α, or other cytokines. The present compositions arealso effective in alleviating other cytokine induced damage includingwasting or cachexia associated with AIDS, tuberculosis, neoplasia ortrauma, and may prevent or ameliorate other disease states associatedwith the production of cytokines, including parasitic infections.

The compositions of the present invention may also be effective in thetreatment of certain chronic degenerative diseases including arthritisand other autoimmune afflictions which are characterized by productionof TNF-α. In this connection, the compositions of the present inventionare contemplated as therapeutically effective in the treatment ofmultiple sclerosis.

The present invention relates to pharmaceutical compositions for thepurposes set out above, in which the active ingredient is a compound ofthe general formula (I):

having the (3S,4S) configuration and being essentially free of the(3R,4R) enantiomer,

wherein A - - - - - B indicates an optional 1(2) or 6(1) double bond,

R is

(a) —Q wherein Q is a heterocyclic moiety having a labile hydrogen atomso that said moiety acts as a carboxylic acid analogue,

(b) —R′X wherein R′ is C₁-C₅ alkyl and X is halogen, —OR″ wherein R″ ishydrogen, C₁-C₅ alkyl, or —OC(O)R′″ wherein R′″ is hydrogen or C₁-C₅alkyl,

(c) —R′N(R″)₂ wherein R′ is C₁-C₅ alkyl and each R″, which may be thesame or different, is hydrogen or C₁-C₅ alkyl optionally containing aterminal —OR′″ or —OC(O)R′″ moiety wherein R′″ is hydrogen or C₁-C₅alkyl,

(d) —R′ wherein R′ is C₂-C₅ alkyl,

(e) —R′OR′″ wherein R′ is C₁-C₅ alkyl and R′″ is hydrogen or C₁-C₅alkyl, or (f) —R′—C(O)OR′″, wherein R′ may be absent and R′ and R′″ areas defined above;

G is (a) halogen, (b) C₁ -C₅ alkyl, or (c) —OR₁ wherein R₁ is (a′) —R″,wherein R″ is hydrogen or C₁-C₅ alkyl optionally containing a terminal—OR′″ or —OC(O)R′″ moiety wherein R′″ is hydrogen or C₁-C₅ alkyl, or(b′) —C(O)R′″ wherein R′″ is as previously defined; and

R₂ is (a) C₁-C₁₂ alkyl, (b) —OR″″, in which R″″ is a straight chain orbranched C₂-C₉ alkyl which may be substituted at the terminal carbonatom by a phenyl group, or (c) —(CH₂)_(n)OR′″ wherein n is an integer of1 to 7 and R′″ is hydrogen or C₁-C₅ alkyl.

In a currently preferred group of compounds, R² designates a1,1-dimethylalkyl radical or a 1,2-dimethylalkyl radical with a total ofat least 7 carbon atoms. Also preferred are precursors of suchcompounds. Particularly preferred compounds are those wherein R² is1,1-dimethylheptyl or 1,2-dimethylheptyl. It is these embodiments of R²that are found in THC and its analogues. However, for thecytokine-inhibiting activity that characterizes the present invention,it is believed that any lower or mid-range alkyl substituent will besuitable at this position.

A preferred compound, with which many of the physiological experimentshave been carried out, is the compound which may be referred to as the(+)-(3S,4S)-1,1-dimethylheptyl homologue of7-hydroxy-Δ⁶-tetrahydrocannabinol. Said compound is designatedhereinafter as HU-211, or by the trivial chemical name dexanabinol.

It is stressed that all the compounds are of the (+)-(3S,4S)configuration, essentially free of the (−)-(3R,4R) enantiomer, thelatter known to possess the undesired psychotropic side-effects. Thus,for example, the enantiomers of the synthetic cannabinoid7-hydroxy-Δ⁶-tetrahydrocannabinol 1,1-dimethylheptyl homologue, havebeen described [Mechoulam, R., et al., Tetrahedron:Asymmetry 1: 315-319,1990; Mechoulam, R. et al., Experientia 44: 762-764, 1988]. The(−)-(3R,4R) enantiomer, herein designated HU-210, is a highly potentcannabimimetic compound (nearly 100 times more active thanΔ-1-tetrahydrocannabinol, the active component of hashish). The(+)-(3S,4S) enantiomer, herein designated HU-211, while known to beactive as a neuroprotective agent, an analgesic and as an anti-emetic,is inactive as a cannabimimetic even at doses several thousand timeshigher than the ED₅₀ of HU-210 (Mechoulam, R. et al., Experientia 44:762-764, 1988).

Another family of stereospecific compounds that falls within the scopeof the present invention is the family of analogs comprising(3S,4S)-delta-6-tetrahydrocannabinol-7-oic acids and derivativesthereof, in which R in formula 1 bears a carboxylic acid, as exemplifiedby the prototype compound HU-245. HU-245 and its analogs are disclosedin international patent application WO 93/05031 which is herebyincorporated in its entirety by reference.

As mentioned above, the compounds of the general formula (I) as definedherein are substantially devoid of cannabimimetic central nervous systemactivity.

Pharmacology

The novel compositions contain in addition to the active ingredientconventional pharmaceutically acceptable carriers, diluents and thelike. Solid compositions for oral administration such as tablets, pills,capsules or the like may be prepared by mixing the active ingredientwith conventional, pharmaceutically acceptable ingredients such as cornstarch, lactose, sucrose, sorbitol, talc, stearic acid, magnesiumstearate, dicalcium phosphate and gums with pharmaceutically acceptablediluents. The tablets or pills can be coated or otherwise compoundedwith pharmaceutically acceptable materials known in the art to provide adosage form affording prolonged action or sustained release. Other solidcompositions can be prepared as suppositories, for rectaladministration. Liquid forms may be prepared for oral administration orfor injection, the term including subcutaneous, transdermal,intravenous, intrathecal, and other parenteral routes of administration.The liquid compositions include aqueous solutions, with or withoutorganic cosolvents, aqueous or oil suspensions, flavored emulsions withedible oils, as well as elixirs and similar pharmaceutical vehicles. Inaddition, the compositions of the present invention may be formed asaerosols, for intranasal and like admiration.

The active dose for humans is generally in the range of from 0.05 mg toabout 50 mg per kg body weight, in a regimen of 1-4 times a day, or byconstant infusion. However, administration every two days or even lessfrequently may also be possible, as the drug has a rather prolongedaction. The preferred range of dosage is from 0.1 mg to about 25 mg perkg body weight. However, it is evident to one sidled in the art thatdosages would be determined by the attending physician, according to thedisease to be treated, method of administration, patient's age, weight,contraindications and the like.

All the compounds defined above are inhibitors of tumor necrosis factoralpha (TNF-α), and can be used as active ingredients of pharmaceuticalcompositions for treatment of one, or simultaneously several, symptomsof the disorders defined above. The effective dosages are essentiallysimilar, and the more pronounced effect is that of inhibition of TNF-αproduction, in addition to the known characteristics of these compounds.However, it is important to note that the compounds and compositions ofthe present invention may exert their beneficial effects by othermechanisms, and specifically may prevent the action of TNF-α indirectlyvia their induction of and/or action on other cytokines. For example,the compositions of the present invention can prevent, or at leastalleviate, IL-1 and IL-6 production, as well as poisoning caused bynitric oxide.

The compounds of the present invention are administered for the abovedefined purposes in conventional pharmaceutical forms, with the requiredsolvents, diluents, excipients, etc. to produce a physiologicallyacceptable formulation. They can be administered by any of theconventional routes of administration. The required dose for humansranges from 0.005 mg/kg to about 50 mg/kg per unit dosage form. The mostpreferred unit dose range is from about 0.1 mg/kg to about 20 mg/kg bodyweight.

It will be appreciated that the most appropriate administration of thepharmaceutical compositions of the present invention will depend on thetype of injury or disease being treated. Thus, the treatment of septicshock will necessitate systemic administration of the drug as rapidly aspossible after diagnosis of the condition. On the other hand, diminutionor prophylaxis of chronic degenerative damage will necessitate asustained dosage regimen.

HU-211conveys significant protection in different models of septic shockassociated with bacterial infection or in models of traumatic shock.

The invention also relates to methods of treatment of the variouspathological conditions described above, by administering to a patient atherapeutically effective amount of the compositions of the presentinvention. The term administration as used herein encompasses oral,parenteral, intravenous, intramuscular, subcutaneous, transdermalintrathecal, rectal and intranasal administration.

It was shown previously that the pharmacological profile of HU-211, andsome other compounds constituting the active ingredients of the presentcompositions, includes the induction of stereotypy and locomotorhyperactivity (Feigenbaum et al., 1989 ibid.). These effects wereobserved at doses far exceeding those contemplated for use in connectionwith repression of cytokines. Indeed Phase I clinical trials of HU-211,administered intravenously to healthy human volunteers showed noevidence of toxicity or psychoactivity of these compounds within thedose ranges contemplated for use in humans.

Test Systems

Evaluation of the therapeutic effects of HU-211and its analogs has nowbeen carried out in a series of experimental systems of increasingsophistication to support the utility of these drugs as inhibitors ofTNF-α.

The TNF inhibitory and/or protective effects have been evaluated both invitro and in vivo. These beneficial protective effects have beencorroborated in the following systems:

(a) Lowering TNF production in rat brain after LPS administration:

The administration of LPS to experimental animals triggers thebiosynthesis of large quantities of TNF. After binding to its receptor,TNF induces a wide variety of cellular responses, which are implicatedin the pathogenesis of septic shock. Rats injected with LPS into theirbrain (intra cerebral-ventricular, icv, injection) serve as a model forendotoxic shock, and the ability of compounds to inhibit the productionin the brain of any of the mediators (e.g. TNF, interleukins) which areactivated in response to LPS is a measure of their potency asanti-endotoxic agents.

(b) Reduced TNF levels in the serum of mice after LPS administration:

An early response to LPS administration is the elevation of serum levelsof TNF, which peaks at 1.5-2 hr after LPS administration. Mice treatedwith LPS serve as a model for the effect of drugs on reducing TNF levelsunder these conditions.

(c) Reduced LPS-induced hypotension in the rat:

A prominent manifestation of endotoxemia is hypotension, and the effectof a drug on mean arterial blood pressure (MABP) indicates its potencyas an anti-endotoxic shock agent. The protective effect of a testcompound is evaluated in rats treated with LPS, in which the bloodpressure and other vital signs are monitored up to 4 hrs after inductionof endotoxemia.

(d) Improved clinical outcome after closed head injury in rats:

Closed head injury is associated with high mortality and morbidity. Itinduces edema formation, blood-brain-barrier (BBB) disruption, neuronalcell death and impairment of motor and cognitive functions. Animalssubjected to head trauma in a controlled fashion serve as models inwhich to test drugs of therapeutic potential. Test compounds can beevaluated both for improved clinical outcome and for reduction of edemainduced by closed head injury. The ability of compounds to reduce theseverity of neurological symptoms and to reduce brain edema isconsidered a measure of their potency in reducing brain damage.

(e) Improved clinical outcome in experimental autoimmune diseases inrats:

Autoimmune diseases are associated with elevated levels of TNF. Themodels which are most conveniently studied are experimental allergicencephalomyelitis (EAE) and experimental autoimmune arthritis inrodents. EAE is an autoimmune neurological disease elicited bysensitization of the animals to myelin basic protein from the centralnervous system, which is also known as basic encephalitogenic protein.EAE is considered by many to represent a model of the human diseasemultiple sclerosis. Experimental autoimmune arthritis is induced inanimals by immunization with collagen in complete Freund's adjuvant Theability of compounds of the general formula I to prevent or attenuatethe clinical symptoms of these autoimmune diseases was tested.

Each of these systems represents an aspect of endotoxin toxicity whichis amenable to intervention by pharmaceutical agents. It is likely thatthe compounds of the present invention exert their demonstratedprotective effects by virtue of their ability to prevent the productionof TNF-α. Nevertheless, it cannot be ruled out that their activity ismediated by other cytokines or additional mechanisms.

The prototype drug used for evaluation of prevention of release of TNF-αis the compound pentoxifylline, a known inhibitor of TNF release, and wehave evaluated the similarities and differences between the biologicalactivities of this compound and HU-211, as summarized in Table 1.

This evaluation clearly supports the concept that HU-211 is not actingsolely by blocking the release of tumor necrosis factor. Rather thetherapeutic effects of HU-211 may be attributable to additionalmechanisms including suppression of TNF production or blockade of TNFaction, among others.

Importantly, it has been shown that the activity of these compounds isstereospecific and that the psychotropic (−) compounds are of loweractivity or even not active in these systems. Thus, HU-210 is noteffective in preventing the action of TNF in vivo. This supports theconcept that the suppression of TNF is not the known immuno-modulatoryactivity, such as has been described for a variety of psychoactiveenantiomers of cannabinoids and analogs (Lynn and Herkenham, J.Pharmacol. and Exp. Ther., 286, 1612-1623, 1993). Even if the (−)compounds were as effective as the (+) compounds of the presentinvention the severe psychotropic activity of the former would maketheir medical use prohibitive.

Compounds

Experiments have shown that the (+)-(3S,4S) stereoisomer of the compoundof formula I wherein A - - - - - B designates a 6(1) double bond, R ismethyl, G is OH, and R² is 1,1-dimethylheptyl and the compound offormula I wherein A-----B designates a 1(2) double bond, R is methyl, Gis OH, and R² is 1,1-dimethylheptyl, both said compounds beingessentially free of the (−)-(3R,4R) enantiomer, have practically thesame activity as that of the compound designated HU-211. The formercompound is designated compound Vb in U.S. Pat. No. 4,179,517; thelatter compound is compound XIb therein.

In addition it has been found that some novel compounds of generalformula (I), wherein R designates CH₂OR′ and R′ designates an acyl groupalso have the desired activity. These novel compounds may be prepared,for example, by esterification of compounds of general formula (I)wherein R designates CH₂OH and G is OH, under conditions which favor theformation of the desired monoesters with relatively high yield.

Among the novel compounds tested, monoesters including nicotinate,succinate and maleate are preferred. Most preferred compounds includethe glycinate ester and N-substituted glycinate ester salts, such as thetrimethyl- and triethylammonium acetate derivatives of HU-211. Thesenovel compounds have the added advantage of being soluble in someaqueous solutions, whereas the parent compounds are extremelyhydrophobic.

The highly lipophilic character of HU-211 enables the compound to enterthe central nervous system, as it readily passes the blood-brainbarrier. However, the high lipid solubility is also associated with verypoor solubility in water. This makes the development of formulationssuitable for intravenous administration of HU-211 difficult, hamperingits clinical application. Water-soluble derivatives of HU-211 designedto readily release the drug by hydrolysis following i.v. administrationmight overcome this problem and will be used as prodrugs. On the otherhand, if the obtained derivatives are hydrolytically stable but possessintrinsic biological activity they could be used as easy-to-formulateanalogs (congeners) possessing significant NMDA antagonist activity.

The novel derivatives are obtained by the attachment of polar orpermanent charge bearing groups to the allylic (C-7) or phenolic (C-3′)hydroxylic functionalities of HU-211, the two sites suitable forreversible structural modification, through a carboxylic ester orphosphate linkage.

EXAMPLES

TNF is released by mononuclear phagocytes and from astrocytes inresponse to infection or endotoxin. It has been implied that TNFmediates the lethal effect of endotoxic shock. In the next examples wedescribe the use of LPS as a model for endotoxic shock in rats and miceand the effect of HU-211 on a) TNF production in response to LPSchallenge, and b) on some physiological manifestations of LPS toxicity.The following examples are intended to illustrate the present inventionand these are to be construed in a non-limitative manner.

Physiological Example 1

The effect of HU-211 on Rat Brain Production of TNF After LPSAdministration

The biological activity (bioassay) of TNF in extracts prepared from ratbrains following intracerebral injection of LPS was assessed. LPSadministration triggers the production of TNF and the effect of HU-211,and other analogs administered 15 min before LPS was evaluated 2 hrlater.

HU-211 and HU-245 significantly attenuated the increase in biologicalactivity of TNF in homogenates prepared from brains of LPS treated rats.

Experimental Procedure

LPS (50 ug, dissolved in 20 ul saline) was injected into the cerebrallateral ventricle and within 5 min of injection, HU-211 7.5 mg/kg iv wasadministered. Two hours later, the rats were decapitated, their brainsremoved, and segments of the cortical tissue taken for cytokinesextraction in DMEM (Dulbecco's modified Eagle's medium), and analysis ofbiological activity using bioassay (Shohami et. al., J. Cereb. BloodFlow Metabol., 14, 615-619, 1994).

Results

The activity of TNF (expressed as S₅₀, see Shohami et al. ibid., fordetails) in control (LPS) treated rats was 407±172 whereas in theHU-211-treated rats it reached only 43±21. These results clearlydemonstrate that HU-211 reduced the biological activity of TNF in thebrain homogenates. In contrast, the (−) enantiomer, which is denotedHU-210 did not affect TNF levels when administered to rats under similarconditions (S₅₀=660±267 in LPS treated rats vs. 783±410 in theLPS+HU-210 treated rats). This establishes the stereospecificty of theeffect of the non-psychotropic (+) enantiomer.

The evaluation of analogs was extended to include rats treated withHU-245 at a dose of 5 mg/kg. Upon bioassay of TNF, these animals werefound to have a S₅₀ of 89±36, vs. 448±146 found for the LPS controlanimals.

The results obtained in the bioassay of TNF activity were confirmed byELISA.

Conclusion

One of the typical host responses to LPS is increased production andactivity of TNF, which is implicated in the pathophysiology of endotoxicshock. The present results demonstrate that HU-211, but not itsenantiomer HU-210, may reduce this local response (LPS was injecteddirectly into the brain, where the TNF production was assessed). HU-245treated animals also had a significantly lowered level of TNF activity.

Physiological Example 2

The Effect of HU-211 on Serum Levels and Biological Activity of TNF inLPS Treated Mice

The following experiment was designed to assess the peripheral effect ofHU-211 on serum levels of TNF in LPS treated mice.

Serum levels of TNF peak within 1.5 hr following LPS administration, andat this time point, we assessed both TNF activity and levels in theserum.

HU-211 and HU-245, when coadministered with LPS, significantly reducedboth levels and activity of TNF in the serum of mice.

Determination of TNF Activity: Experimental Procedure

Mice (C57BL/6)were injected ip with 100 μg LPS, concomitantly withHU-211, HU-210, or HU-245 (200 μg/mouse), and 90 min later they werebled, the sera separated and TNF was assayed for its activity (bioassay)and levels (using ELISA kit).

Results

FIG. 1 (A&B) depicts the activity (S50) and levels (pg/ml) of TNF in thesera of the LPS treated mice. It is apparent that HU-211 attenuated theLPS-induced production and action of TNF to a very pronounced extent.

HU-245 also showed a pronounced effect on TNF levels, as determined bybioassay. The S₅₀ found for animals treated with HU-245 was 44±11 vs.1233±237 found for control animals treated with LPS alone.

Physiological Example 3

Protective Effect of HU-211 on Survival of LPS Treated Mice

The effect of HU-211 on the survival of mice, in response toadministration of LPS was assessed. The survival was followed up to 48hr.

HU-211, administered 30 min prior to LPS, significantly reducedmortality of the mice.

Animals and Materials

BALB/c male mice 20-25 gr (Anilab, Israel); HU-211 5% in cosolvent,(Pharmos Ltd., Israel, batch no. 4T01HS); Blank cosolvent, (Pharmosbatch no. c-00-502002); LPS, E. Coli 055:B5, (Difco USA, batch no.1001007); Saline (Braun, Germany batch no. 3431A41A).

Procedure

Animals are administered with either HU-211 at a dose of 10 mg/kg i.p.or vehicle alone (10 ml/kg i.p.), and 30 minutes later with LPS IV. Twodose levels of LPS were tested in the present study: 10 and 15 mg/kg.

Mortality rate was recorded once a day for 2 days.

Results (FIG. 2A)

LPS 15 mg/kg dose level caused 67% mortality (4/6, survival rate of 33%)24 hours post vehicle administration. By 48 hours post vehicleadministration none of the vehicle treated mice survived.

LPS 15 mg/kg dose level caused 33% mortality (2/6, 67% survival rate) 24hours post HU-211 administration. By 48 hours post HU-211 administrationnone of the animals survived. The difference between HU-211 activity andvehicle was not statistically significant, at this dose level.

LPS 10 mg/kg dose level caused 57% mortality (8/14, 43% survival rate)24 hours post vehicle administration. At 48 hours after vehicleadministration none of the animals survived.

The same dose level caused only 9% mortality (1/12, 91% survival rate)24 hours post HU-211 administration. After 48 hours 4 out of the 12tested animals survived (33%). None of the animals survived 72 hourspost LPS administration. These differences were statisticallysignificant, p<0.02 Fisher exact test for both 24 and 48 hours.

Conclusions

HU-211 at a dose level of 10 mg/kg i.p. administered 30 minutes beforeLPS administration, induced a statistically significant protection(p<0.02) at both 24 and 48 hrs. after LPS administration.

Physiological Example 4

Protection Against LPS-induced Hypotension

The purpose of this study was to test the possible effect of HU-211 onthe cardiovascular alterations induced by LPS administration.

Animals and Materials

Male and female Sprague Dawley rats (Harlan, Israel); HU-211 5% incosolvent, (Pharmos Ltd., Israel, batch no. 4T01HS); Blank cosolvent,(Pharmos batch no. c-00-502002); LPS, E. Coli 055:B5, (Difco USA, batchno. L001007); Saline (Braun, Germany batch no. 3431A41A); Halothaneanesthetic (Fluthane, Zeneca, U.K. batch no. ML040); Heparin 5000 IU/ml(Chaoy, France batch no. 1842).

Procedure

Rats were individually anesthetized with halothane in 70% nitrogen, 30%oxygen. The right femoral artery was catheterized using polyethylenetubes (PE-50, Clay Adams, USA), and connected to a computerizedphysiograph through a pressure transducer (XTT Vigo, USA). The animalsare then connected via a rectal probe to the above physiologicalrecorder. Rectal temperature is kept throughout the study between 37° C.and 38° C., using a heating lamp. Following 10-15 minutes of baselinerecording, animals are intravenously administered an injection ofsaline, vehicle alone or HU-211 at a dose of 4 mg/kg. As soon aspossible after completion of the first injection (2-5 minutes later)they are administered an intravenous injection of LPS at a dose of 15mg/kg (in a volume of 3 ml/kg). Blood pressure and heart rate arerecorded for the following 4 hrs. The hematocrit is measured andrecorded just prior to drug administration, and 1, 2, and 3 hours later.At the end of the follow up period, animals are sacrificed by deepanesthesia with halothane.

Results

Administration of LPS at a dose of 15 mg/kg to rats caused a reductionof around 30% in mean arterial blood pressure (MABP). This drop in bloodpressure was abolished by pre-administration of HU-211 (at a dose of 4mg/kg) intravenously. A transient hypotension was evident 10 minutesafter LPS administration in the HU-211 treated rats. This lasted for5-10 minutes and by 30 minutes post-LPS administration normotension wasregained. The differences in MABP were statistically significant betweenthe HU-211 treated and the control groups (Student's t-test), as shownin FIG. 3a. LPS also induced an increase of about 10% in hematocrit.This increase was prevented by administration of HU-211 at a dose of 44mg/ml administered intravenously 2-3 minutes before LPS administration(FIG. 3b). These differences were statistically significant 60 and 120minutes post LPS administration.

Physiological Example 5

Protection Against Septic Shock

The effect of HU-211 in endotoxic shock was assessed in a model oflethal toxicity by lipopolysacharide (LPS) in D-galactosamine(D-GALN)-sensitized mice (Galanos et al. PNAS 76: 5739, 1979, Lehman etal. J. Exp. Med. 165: 657, 1987). In this model 100% mortality occurswithin 6-8 hrs of administration of the combined treatment.

Experimental Procedure

LPS (10 ng/mouse) and D-GALN (18 μg/mouse) were coadministered to 20mice (C57BL/6). Half of the mice served as controls, and the other weretreated within 5 min of injection with HU-211 (7.5 mg/kg body weight),followed by 8 additional injections every hour. The survival wasfollowed up to 24 hrs, and the results of this experiment are depictedin Table 1.

TABLE 1 The effect of HU-211 on mice survival after endotoxic shocklethality in control lethality in HU-211 treated group exp. no group:dead/total dead/total 1  5/5 1/5 2 10/10 1/10 3  5/5 0/5 total: 20/20(0% survival) 2/20 (90% survival)

The treatment with HU-211 rescued 90% of the mice (FIG. 2B), with 18 outof 20 HU-211 treated mice surviving the shock, whereas none of thecontrols (20 mice) survived.

Conclusion

To date, mortality from septic shock remains very high, despite advancesin surgical and medical sciences. The results of our experiment areencouraging and may serve as a basis for saving lives of patients withseptic shock.

Physiological Example 6

The Effect of HU-211 on TNF Production in a Rat Model of Head Trauma

The effect of HU-211 on TNF production was assessed in a model of headtrauma (HT) in rats. Injury was induced in anesthetized rats by aweight-drop device followed by a recovery period of up to 30 days. Thistype of trauma produces brain edema (i.e. increase in water content,decrease in specific gravity in the brain), breakdown of the blood brainbarrier (BBB), clinical and cognitive dysfunction.

HU-211 significantly reduces reduced edema and improved the clinicaloutcome measured 48 hours after HT (Shohami et al., J. Neurotrauma 10:109, 1993; Brain Res. 674, 55-62, 1995)

Experimental Procedure

The model was described in detail by Shapira et al., Crit. Care Med. 16:258-265, 1988. Rats were subjected to head trauma (HT) by a weight-dropdevice and surviving rats were followed up for 30 days post trauma. Inthis experiment we compared the effects of two agents: 1) pentoxyfilline(PTX), a methylxanthine known for its ability to prevent or attenuatethe LPS-induced release of TNF both in vitro and in vivo. This effectwas found to be exerted at the transcriptional level (Doherty et al.,1991); and 2) HU-211, which was previously shown to have potentcerebroprotective effects in the CHI model (Shohami et al. loc. cit.,FIG. 4A). The levels of TNF were measured after CHI in order todetermine whether the beneficial effect of HU-211 is also associatedwith suppression of TNF production or activity.

Results

As shown in FIG. 4, the protective effect of HU-211 is significantlysuperior to that of pentoxyfilline in terms of brain tissue watercontent (a measure of the anti-edema effect of the compounds) and interms of the brain levels of TNF after CHI.

Conclusion

Severe head injury, or cerebral ischemia, is associated with a highmortality rate (exceeding 50%) and poor functional outcome. Despiteextensive clinical and experimental research, there are no well-definedtherapies for these conditions. There are very few available treatmentsfor brain injury today and the gradual progressive biochemical changeswhich occur after head trauma can lead to the evolution of permanentneuronal damage. The results clearly demonstrate that the compounds ofthe instant invention, namely HU-211, possess protective properties in amodel of closed head injury in rats.

Physiological Example 7

Attenuation of Experimental Autoimmune Arthritis in Mice

The purpose of this study is to test the activity of HU-211 vs.arthritis, induced by collagen type 2 incorporated into completeFreund's adjuvant.

Materials and Methods

CD-1 male mice (27-33 gr.), five in each treatment group (supplied byHarlan Israel) were used in the present study. Each animal wasadministered with collagen type 2 (Sigma, batch no. 111H7180) 100 μg/mlin 0.1 ml complete Freund's adjuvant (Sigma batch no. 084H8800). Thecollagen was administered s.c. into the base of the tail. The volume ofeach hind paw was measured using a plathysmometer (Hugo Basill, Italy)before collagen administration and on days 1, 4, 7, 10, 13 and 16 oftreatment (30 minutes after drug treatment).

Five treatment groups were tested in the present study: vehicle (blankcosolvent) 10 ml/kg once daily, HU-211 10 mg/kg every three days, HU-21120 mg/kg once daily, HU-211 20 mg/kg every 3 days and diclofenac 10mg/kg (10 ml/kg) every 3 days. All treatments were administeredintraperitoneally. HU-211 was used from a 5% preparation in cosolvent(Pharmos batch no. PRN-C-08623041), by diluting with saline (B. Braunbatch no. 343A41A) 1:25. The same procedure was performed with blankcosolvent (Pharmos batch no. 608945). Diclofenac was prepared inPharmos, batch no. LG-121/70 1 mg/ml. At the same time paws weremeasured they were clinically evaluated according to the followingmethod (Williams R. O., Proc. Natl. Acad. Sci. USA: 89:9784-9788):0—normal, 1—slight swelling and erythema, 2—pronounced edematousswelling and 3—joint rigidity. On day fifteen of treatment animals wereeuthanised with pentobarbital 100 mg/kg ip. (Pental veterinary, C.T.S.Israel, batch no.170005). Blood samples were taken for hematocrit andblood levels of HU-211 (in heparinized serum).

Results

a. Clinical Signs: The primary arthritis related sign that was evidentin the present study was swelling of the paws. Animals treated withHU-211 20 mg/kg (daily or every 3 days) demonstrated the lowest numberof these signs, compared to the other treatment groups. Thesedifferences were compared using a non parametric analysis (Wilcoxon RankSum Test), and were found to be statistical significant (p<0.01).

b. Paw volume (FIG. 5): All treatments (except the HU-211 20 mg/kg every3 days) reduced the collagen-induced paw edema by 30-50% compared to thevehicle treated animals (up to 30% increase in paw volume in the treatedmice compared to up to 40% increase in the vehicle treated mice). Thiseffect was statistically significant (p<0.05) with HU-211 20 mg/kg(daily treatments) and with the HU-211 10 mg/kg every 3 days. Thediclofenac treated animals demonstrated a smaller paw volume compared tothe vehicle treated rats. But this difference did not reach statisticalsignificance.

Physiological Example 8

Attentuation of Experimental Autoimmune Encephalomyelitis in Lewis Rats

EAE was tested using Lewis rats in which the disease displays onset ofsymptoms around day 10 after induction and spontaneous recovery around18 days after induction of the disease. The Lewis rats used were 8 weekold females (from OLAC, England). The animals (5 per cage) weremaintained on a 12 hour light/12 hour dark regimen, at a constanttemperature of 22° C., with food and water ad libitum. EAE was inducedin these animals by immunization with purified guinea pig myelin basicprotein emulsified in complete Freund's adjuvant. Guinea pig myelinbasic protein (mbp) was prepared from spinal cord homogenates defattedwith chloroform/ethanol and the isolated protein was purified using ionexchange chromatography. Each animal received 50 micrograms of thepurified protein. A solution of mbp (0.5 mg/ml) was emulsified with anequal volume of Complete Freund's Adjuvant containing 4 mg/ml ofmycobacterium tuberculosis, and each animal received 100 microliters (50μl in each hind foot pad).

Animals were treated with a single injection of HU-211 or vehiclecontrol administered intravenously in a volume of 2 ml. The time oftreatment was varied from day 10 to day 18 post induction of disease,with five animals per group. The results of this experiment, which arepresented in FIG. 6, show the diminution of mean clinical score inHU-211 treated animals. (Score of 1 indicates tail paralysis, 2indicates paraplegia, 3 indicates quadriplegia, 4 indicates completebody paralysis and 5 indicates death).

Although the present invention has been described with respect tovarious specific embodiments thereof in order to illustrate it, suchspecifically disclosed embodiments should not be considered limiting.Many other specific embodiments will occur to those skilled in the artbased upon applicants' disclosure herein, and applicants propose to bebound only by the spirit and scope of their invention as defined in theappended claims.

What is claimed is:
 1. A method for septic shock syndrome prophylaxis by preventing production of tumor necrosis factor by administering to a patient in need thereof a pharmaceutical composition comprising a therapeutically effective amount of a compound of the formula (I):

having the (3S,4S) configuration and being essentially free of the (3R,4R) enantiomer, wherein A - - - - - B indicates an optional 1(2) or 6(1) double bond, R is (a) —Q wherein Q is a heterocyclic moiety having a labile hydrogen atom so that said moiety acts as a carboxylic acid analogue, (b) —R′X wherein R′ is C₁-C₅ alkyl and X is halogen, —OR″ wherein R″ is hydrogen, C₁-C₅ alkyl, or —OC(O)R′″ wherein R′″ is hydrogen or C₁-C₅ alkyl, (c) —R′N(R″)₂ wherein R′ is C₁-C₅ alkyl and each R″, which may be the same or different, is hydrogen or C₁-C₅ alkyl optionally containing a terminal —OR′″ or —OC(O)R′″ moiety wherein R′″ is hydrogen or (d) —R′ wherein R′ is C₁-C₅ alkyl, (e) —R′OR′″ wherein R′ is C₁-C₅ alkyl and R′″ is hydrogen or C₁-C₅ alkyl, or, (f) —R′—C(O)—OR″′, wherein R′ may be absent and R′ and R″′ are as defined above; G is (a) halogen, (b) C₁-C₅ alkyl, or (c) —OR₁ wherein R₁ is (a′) —R″, wherein R″ is hydrogen or C₁-C₅ alkyl optionally containing a terminal —OR′″ or —OC(O)R′″ moiety wherein R′″ is hydrogen or C₁-C₅ alkyl, or (b′) —C(O)R′″ wherein R′″ is as previously defined; and R₂ is (a) C₁-C₁₂ alkyl, (b) —OR″″, in which R″″ is a straight chain or branched C₂-C₉ alkyl which may be substituted at the terminal carbon atom by a phenyl group, or (c) —(CH₂)_(n)OR′″ wherein n is an integer of 1 to 7 and R′″ is hydrogen or C₁-C₅ alkyl.
 2. The method of claim 1 wherein A - - - - - B designates a 1(2) or 6(1) double bond, R is lower alkyl of 1-3 carbon atoms or CH₂OH, G is hydroxy or a lower acyloxy group, and R² is (a) a straight or branched C₆-C₁₂ alkyl radical or (b) a group OR³ in which R³ is a straight or branched C₅-C₉ alkyl radical optionally substituted at the terminal carbon atom by a phenyl group.
 3. The method of claim 1 wherein A - - - - - B designates a 1(2) or 6(1) double bond, R is COOR″′ wherein R″′ is lower alkyl of 1-5 carbon atoms or hydrogen, G is hydroxy or a lower acyloxy group, and R² is (a) a straight or branched C₆-C₁₂ alkyl radical or (b) a group OR³ in which R³ is a straight or branched C₅-C₉ alkyl radical optionally substituted at the terminal carbon atom by a phenyl group.
 4. A method for septic shock syndrome prophylaxis by blocking the action of tumor necrosis factor in a patient which comprises administering to said patient, in a manner calculated to block said cytokine in a stereospecific manner, a therapeutically effective amount of a compound of the formula (1):

having the (3S,4S) configuration and being essentially free of the (3R,4R) enantiomer, wherein A - - - - - B indicates an optional 1(2) or 6(1) double bond, R is (a) —Q wherein Q is a heterocyclic moiety having a labile hydrogen atom so that said moiety acts as a carboxylic acid analogue, (b) —R′X wherein R′ is C₁-C₅ alkyl and X is halogen, —OR″ wherein R″ is C₁-C₅ alkyl, or —OC(O)R′″ wherein R′″ is hydrogen or C₁-C₅ alkyl, (c) —R′N(R″)₂ wherein R′ is C₁-C₅ alkyl and each R″, which may be the same or different, is hydrogen or C₁-C₅ alkyl optionally containing a terminal —OR′″ or —OC(O)R′″ moiety wherein R′″ is hydrogen or C₁-C₅ alkyl, (d) —R′ wherein R′ is C₁-C₅ alkyl, (e) —R′OR′″ wherein R′ is C₁-C₅ alkyl and R′″ is hydrogen or C₁-C₅ alkyl, or, (f) —R′—C(O)—OR″′, wherein R′ may be absent and R′ and R″′ are as defined above; G is (a) halogen, (b) C₁-C₅ alkyl, or (c) —OR₁ wherein R₁ is (a′) —R″, wherein R″ is hydrogen or C₁-C₅ alkyl optionally containing a terminal —OR′″ or —OC(O)R′″ moiety wherein R′″ is hydrogen or C₁-C₅ alkyl, or (b′) —C(O)R′″ wherein R′″ is as previously defined, and R₂ is (a) C₁-C₁₂ alkyl, (b) —OR″″, in which R″″ is a straight chain or branched C₂-C₉ alkyl which may be substituted at the terminal carbon atom by a phenyl group, or (c) —(CH₂)_(n)OR′″ wherein n is an integer of 1 to 7 and R′″ is hydrogen or C₁-C₅ alkyl.
 5. A method as in claim 4 wherein A - - - - - B designates a 1(2) or 6(1) double bond, R designates a —CH₃ or CH₂OH, G designates hydroxy or a lower acyloxy group and R² designates (A) a straight or branched C₆-C₁₂ alkyl radical; (B) a group —O—R³, in which R³ is a straight or branched C₅-C₉ alkyl radical optionally substituted at the terminal carbon atom by a phenyl group.
 6. A method as in claim 4 wherein A - - - - - B designates a 1(2) or 6(1) double bond, R designates a —COOR″′, wherein R″′ is a lower alkyl group of 1-5 carbon atoms or hydrogen, G designates hydroxy or a lower acyloxy group and R² designates (A) a straight or branched C₆-C₁₂ alkyl radical; (B) a group —O—R³, in which R³ is a straight or branched C₅-C₉ alkyl radical optionally substituted at the terminal carbon atom by a phenyl group.
 7. A method for preventing production of tumor necrosis factor by administering to a patient who exhibits the symptoms associated with autoimmune diseases that result from the production of elevated levels of tumor necrosis factor alpha, a pharmaceutical composition comprising a therapeutically effective amount of a compound of the formula (I):

having the (3S,4S) configuration and being essentially free of the (3R,4R) enantiomer, wherein A - - - - - B indicates an optional 1(2) or 6(1) double bond, R is (a) —Q wherein Q is a heterocyclic moiety having a labile hydrogen atom so that said moiety acts as a carboxylic acid analogue, (b) —R′X wherein R′ is C₁-C₅ alkyl and X is halogen, —OR″ wherein R″ is hydrogen, C₁-C₅ alkyl, or —OC(OR′″ wherein R′″ is hydrogen or C₁-C₅ alkyl, (c) —R′N(R″)₂ wherein R′ is C₁-C₅ alkyl and each R″, which may be the same or different, is hydrogen or C₁-C₅ alkyl optionally containing a terminal —OR′″ or —OC(O)R′″ moiety wherein R′″ is hydrogen or C₁-C₅ alkyl, (d) —R′ wherein R′ is C₁-C₅ alkyl, (e) —R′OR′″ wherein R′ is C₁-C₅ alkyl and R′″ is hydrogen or C₁-C₅ alkyl, or, (f) —R′—C(O)—OR′″, wherein R′ may be absent and R′ and R′″ are as defined above; G is (a) halogen, (b) C₁-C₅ alkyl, or (c) —OR₁ wherein R₁ is (a′) —R″, wherein R″ is hydrogen or C₁-C₅ alkyl optionally containing a terminal —OR′″ or —OC(O)R′″ moiety wherein R′″ is hydrogen or C₁-C₅ alkyl, or (b′) —C(O)R′″ wherein R′″ is as previously defined; R₂ is (a) C₁-C₁₂ alkyl, (b) —OR″″, in which R″″ is a straight chain or branched C₂-C₉ alkyl which may be substituted at the terminal carbon atom by a phenyl group, or (c) —(CH₂)_(n)OR′″ wherein n is an integer of 1 to 7 and R′″ is hydrogen or C₁-C₅ alkyl, wherein the compound blocks the action of tumor necrosis factor.
 8. A method according to claim 1 or claim 4 in which said pharmaceutical composition includes a carrier or diluent.
 9. The method of claim 8 which comprises selecting the carrier or diluent to be an aqueous cosolvent solution comprising a pharmaceutically acceptable cosolvent, a micellar solution prepared with natural or synthetic ionic or non-ionic surfactants, or a combination of such cosolvent and micellar solutions.
 10. The method of claim 8 which comprises selecting the carrier to be a solution of ethanol, a surfactant, and water.
 11. The method of claim 8 which comprises selecting the carrier to be an emulsion comprising a triglycerides, lecithin, glycerol, an emulsifier, an antioxidant, and water.
 12. A method according to claim 1 or claim 4 wherein the daily dosage of said compound is between 0.1 and 25 mg/kg.
 13. A method according to claim 1 or claim 4, in which said pharmaceutical composition is administered intravenously, intrathecally, orally or by inhalation. 